The best method for Diagnosis of Cutaneous Leishmaniasis and identification of the causative Leishmania species in Al-Najaf governorate by using PCR assay

Background: The diagnosis of cutaneous leishmaniasis (CL) might be difficult, in particular in endemic areas where different species of Leishmania can cause lesions of very similar appearance and where other skin diseases with similar clinical symptoms occur. Even today, the parasitological diagnosis of CL remains the gold standard and it is based on the direct identification of amastigotes in microscopy smears and/or culture of promastigotes from infected tissues. Although these techniques are highly specific, they are not sensitive enough. Objective: The present study aimed to choose the best method and best medium in isolation and cultivation of cutaneous leishmania parasite that gives rapid recovery of parasite in the first inoculation from the patients and determine the effectiveness of a polymerase chain reaction (PCR) technique for identification of Leishmania species in Al-Najaf governorate by PCR assay and differentiation of the cutaneous leishmania parasites in clinical sample collected from lesion exudates patients. Methods: A total of 138 patients presenting with cutaneous lesions suggestive of CL were sampled for parasitological diagnosis by direct examination (DE), cutaneous leishmania parasites were cultured in tow different media include Novy-Mac Neal-Nicolle (NNN) medium and Roswell Park Memorial Institute medium (RPMI)1640 with fetal calf serum then DNA isolation. The DNA of the promastigote were amplified by PCR including primers selected on repetitive KDNA for identification of leishmania species. The data was analyzed by using frequency and percentage. Results: It's found that NNN medium was the best medium in maintenance and survival growth of parasite and RPMI1640 was the best medium in increasing parasite number gtfas a same peak growth period, which only one day. The recovery time of isolated cutaneous leishmania from RPMI1640 with FCS was the most rapid medium in leishmania parasite isolation (seven days) in comparison with NNN medium (twenty one to thirty days). On other part comparison done between both isolation of cutaneous leishmania parasite and cultivation of an old cultured cutaneous leishmania parasite from NNN media in RPMI1640 with 10% FCS there is difference in growth peak number and in recovery time .The PCR result showed that two species of Leishmania (Leishmania major with 620 bp and Leishmania tropica with 760 bp) exist . Conclusions: : The PCR technique has high specificity and sensitivity during the differentiation between Cutaneous Leishmanial species compared to the conventional methods.